PROJECT SUMMARY Gastrointestinal (GI) motility and defecation are absolute prerequisites for nutrient absorption, fecal elimination and overall health. Normal GI motility, vascular perfusion, and intestinal inflammation are coordinated by vast numbers of neurons that reside within ganglia of the enteric nervous system (ENS) intrinsic to the gut wall. While recent work has identified diverse genes that direct the initial development of progenitor cells that give rise to enteric neurons in the wall of the intestine, we know very little about the genes that are expressed in adult enteric neurons. Consequently we are unable to determine whether efforts to generate enteric neurons produce the normal complement of cell types. Moreover we do not fully understand how distinct types of neurons contribute to overall coordination of intestinal motility because the use of common immunohistochemical markers alone does not distinguish functionally distinct subtypes. As a result, our abilities to target and functionally manipulate specific types of neurons in the gut are extremely limited. To surpass these limitations, our application proposes to develop a comprehensive, single cell transcriptome map of adult enteric neurons in normal mice in parallel with deep sequencing of enteric ganglia from distinct regions of human intestine so that a global gene expression atlas of human enteric ganglia is obtained. To capture mouse enteric neurons for single cell RNA-Seq we will use a fluorescent transgenic mouse line that we developed for live-cell imaging of enteric neurons. Human enteric ganglia will be collected by laser capture microdissection from adult surgical remnants. Comparison of enteric neuron expression profiles between mouse and human data sets will identify conserved genes that mark distinct neuronal subtypes. The resulting expression atlas of enteric neurons will define specific molecular fingerprints for discrete neuron subtypes that are essential to pursue targeted, functional manipulation of GI motility in distinct regions of the intestine.